title: Purification and characterization of a new L-methioninase from solid cultures of Aspergillus flavipes.
abstract: L-Methioninase was purified to electrophoretic homogeneity from cultures of Aspergillus flavipes using anion-exchange and gel filtration chromatography by 12.1 fold compared to the crude enzyme preparation. The purified enzyme had a molecular mass of 47 kDa under denaturing conditions and an isoelectric point of 5.8 with no structural glycosyl residues. The enzyme had optimum activity at pH 7.8 and pH stability from 6.8-8.0 at 35°C. The enzyme appeared to be catalytically stable below 40°C. The enzyme activity was strongly inhibited by DL-propargylglycine, hydroxylamine, PMSF, 2-mercaptoethanol, Hg(+), Cu(2+), and Fe(2+), with slight inhibition by Triton X-(100). A flavipes L-methioninase has a higher catalytic affinity towards L-methionine (Km, 6.5 mM and Kcat, 14.1 S(-1)) followed by a relative demethiolating activity to L-homo-cysteine (Km, 12 mM and Kcat, 9.3 S(-1)). The enzyme has two absorption maxima at 280 and 420 nm, typical of other PLP-enzymes. Apo-L-methioninase has the ability to reconstitute its structural catalytic state completely upon addition of 0.15 mM PLP. L-Methioninase has neither an appreciable effect on liver function, platelet aggregation, nor hemolysis of human blood. The purified L-methioninase from solid cultures of A. flavipes displayed unique biochemical and catalytic properties over the currently applied Pseudomonad enzyme.
authors.affiliation: Microbiology Department, Faculty of Science, Zagazig University, Zagazig, 44519, Egypt. ash_elsayed25@yahoo.com
authors.forename: Ashraf S A
authors.initials: AS
authors.lastname: El-Sayed
mesh_terms: D000818:Animals; D001230:Aspergillus / Q000201:enzymology*; D013437:Carbon-Sulfur Lyases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism* / Q000633:toxicity; D002449:Cell Aggregation; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D006461:Hemolysis; D006710:Homocysteine / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008111:Liver Function Tests; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D006710:Homocysteine; D008715:Methionine; D013437:Carbon-Sulfur Lyases; C021558:L-methionine gamma-lyase